Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
The Andor iQ system employs what we call "active blanking."
The EMCCD and our AOTF communicate directly with each other so that the
AOTF only passes excitation light while the camera is exposing. This
great reduces effects of phototoxicity due to exposure of cells to
excitation light between captures.
Best,
Gary Laevsky, Ph.D.
Imaging Application Specialist
Andor Technology
discover new ways of seeing
[log in to unmask]
Cell (774) 291 - 9992
Office (860) 290 - 9211 x219
Fax (860) 290 - 9566
Web: www.andor.com
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Nowell, Cameron
Sent: Wednesday, November 28, 2007 2:51 AM
To: [log in to unmask]
Subject: Re: Help with EMCCD evaluation
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Neville,
While an EMCCD camera will decrease your exposure time,
you may find that it will not really help solve your phototoxicity
problem. The time your cells are exposed to fluorecent light will not
neccesarily be dependant on the exposure time of your camera, but on how
fast your entire sytem (microscope, shutters, filters, etc.) shuffles
everything around.
What software etc are you using to capture your images?
We have a wide filed live cell rig here that uses an Orca ER camera on
an IX81 microscope and it is all driven by metamorph. Even when the
camera is capturing at 100ms exposures, the samples are exposed to
flourecent light for about 2 seconds. To get rid of these sort of delays
you need to move to using filter wheels, or for even shorter exposuers
something like olympus' CellR system
Cheers
Cam
Cameron Nowell B. Sc (Hons)
Microscopy Imaging and Research Core Facility
Peter MacCallum Cancer Centre
7 St Andrews Place
East Melbourne, Victoria 3002
AUSTRALIA
Phone: +61396561242
Mobile: +61422882700
Fax: +61396561411
________________________________
From: Confocal Microscopy List on behalf of Neville Sanjana
Sent: Wed 28/11/2007 7:57 AM
To: [log in to unmask]
Subject: Help with EMCCD evaluation
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi,
I am in the market for an EMCCD for widefield fluorescence
time-lapse imaging of genetically-labeled cultured cells. I
am acquiring images frequently (every 10 minutes) for
several days. The cells seem to be very sensitive to
phototoxicity and photobleaching (GFP is the fluorophore),
so I am hoping that replacing my Roper CoolSnapHQ CCD with
an EMCCD will allow me to use much shorter exposure times.
I realize that this is a confocal list and my application is
widefield but since EMCCDs are also used with spinning disk
confocal, I thought I'd ask. Apologies if this question has
been hashed out before... I searched the archives without
finding definitive recommendations. (Maybe that's asking for
too much!) Any help on the following would be much appreciated:
1) What EMCCDs have people found to be the most *sensitive*
(nice picture in low-light) and the most *reliable* (driver
doesn't crash, etc.)? I'd especially be interested in any
experiences or opinions with Roper/Photometrics's CascadeII
or QuantEM and the Andor iXon cameras.
2) How have people been testing cameras? I've set up demos
of several cameras so I'd like to do some standard tests.
I've seen some references on the web for calculating dark
noise, read noise, and gain, such as:
http://www.qsimaging.com/ccd_noise_measure.html
http://www.mirametrics.com/tech_note_ccdgain.htm
http://www.photomet.com/library/library_encyclopedia/library_enc_gain.ph
p
but I'd love to hear what other tests people think are
relevant for these cameras. Obviously, I'll try imaging my
samples (using fiber-coupled LED as a constant illumination
source)... what else should I do? Fluorescent beads instead
of my cells perhaps?
3) For coupling the camera to my Olympus IX71, is there
something like the Zeiss Optovar (which, as I understand it,
has multiple selectable magnifications of the intermediate
lens) made by a third party? This would be helpful for
changing between achieving the full NA of the objective and
getting less resolution but a bigger field of view for
different experiments.
Thanks for your help. Best,
- Neville
---
Neville Sanjana
Dept. of Brain and Cognitive Science
Massachusetts Institute of Technology
This email (including any attachments) may contain
confidential and/or legally privileged information and is
intended only to be read or used by the addressee. If you
are not the intended addressee, any use, distribution,
disclosure or copying of this email is strictly
prohibited.
Confidentiality and legal privilege attached to this email
(including any attachments) are not waived or lost by
reason of its mistaken delivery to you.
If you have received this email in error, please delete it
and notify us immediately by telephone or email. Peter
MacCallum Cancer Centre provides no guarantee that this
transmission is free of virus or that it has not been
intercepted or altered and will not be liable for any delay
in its receipt.
|
